Wells coated with HA-binding protein were incubated with serum samples diluted in reaction buffer for one hour and were then discarded.
For in vitro binding assays, recombinant proteins were incubated together for one hour at 4°C and washed extensively prior to analysis by SDS-PAGE and coomassie staining.
In radioactive binding assays, 35S-labelled proteins were incubated with GST fusion protein-coupled Sepharose beads in binding buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF). Assay performed with GST alone constituted the negative control.
For α-Syn binding to vesicles, 5 μg proteins were incubated with 100 µg vesicles for 2 hr at ambient temperature.
Prior studies with this binding assay indicated that small molecule regulators of binding needed to be present while proteins were incubated to form complexes, as well as in the nondenaturing gels and electrophoresis buffer used to resolve the complexes.
To assess the DNA-binding ability of MJ0927, purified proteins were incubated with ssDNA or dsDNA and the resulting complexes were separated by EMSA.
The nuclear proteins were incubated with 5x binding buffer (Pierce), 1 μg poly (dI-dC), 50,000 cpm of P-labeled oligonucleotide for 30 min at room temperature.
In a Northwestern assay, we electrophorezed purified recombinant GPKOW proteins on SDS PAGE gels; upon renaturation, immobilized proteins were incubated for direct binding with P-labelled RNA (bottom panels).
To further validate our findings, an in vitro microtubule binding assay was performed in which purified proteins were incubated with a preparation of polymerised tubulin prior to ultracentrifugation (Fig. 4C).
Cell lysates containing 20 μg proteins were incubated in Rac-GTP-binding protein-coated 96-well plates.
